Long lived invertebrates are immunoincompetent in the sense that they lack the classical T and B cell arms of the immune system but they respond successfully to challenge with micro-organisms by inflammatory mechanisms involving sensitized phagocytes. This proposal follows from a previous grant (GM 30672) on """"""""animal lectins as recognition molecules"""""""", finding that certain carbohydrate-binding lectins of tunicate species could be identified as members of two families of molecules known to be crucial to immune defense; namely, acute phase proteins of the pentraxin type (e.g. C-reactive proteins) and members of the immunoglobulin superfamily. We focus upon immune-like recognition molecules of tunicates because these protochordate species are ancestral to true vertebrates but are lacking in the sophisticated features of B and T cell immunity. (1) We have devised fractionation schemes for the purification of galactose-binding lectins of the tunicate Didemnum candidum, two lectins specific for sialoconjugates and one for fucose from the tunicate Halocynthia pyriformis, a sialic acid binding lectin from the lamprey and a molecule from the tunicate Boltenia ovipera that is serologically cross-reactive with Igs of lower species and with antibodies to synthetic immunoglobulin joining region peptide. The purified molecules will be characterized physicochemically by determining a) native and subunit molecular weights, b) carbohydrate and amino acid compositions, c) the number and affinity of binding sites, and d) investigations of secondary structure using optical rotatory dispersion and circular dichroism measurements. (2) We will determine the amino acid sequence of these molecules, preparing peptides using proteolytic enzymes of defined specificity and chemical cleavage reagents such as CNBr with the resolution of peptides by gel filtration chromatography using high performance liquid chromatography and by reverse phase peptide chromatography. The peptides will be sequenced using the ABI pulsed liquid phase sequencer. The amino acid sequence will facilitate detailed comparisons with known recognition molecules and enable the synthesis of synthetic oligonucleotide probes to be used in isolation of relevant genes. (3) To ensure that we obtain complete sequence information for the tunicate and lamprey lectins, we will employ the technology of molecular biology. Two strategies will be followed: the first is to produce libraries of genomic DNA and screen these with synthetic oligonucleotide probes predicted from the available sequence of the lectin molecules. A second strategy will be to produce cDNA libraries constructed using total mRNA in expression vectors will then be screened serologically using antibodies to the purified lectins and the synthetic probes. DNA will be sequenced using Sanger's dideoxy chain determination method. We will characterize the genes in terms of their number and types of segments and the arrangements of these elements.
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